Transient expression assays

The genetically complex AcAfNPV prototype baculovirus genome is a double-stranded, covalently closed circular molecule with superhelical conformation and a size of approximately 130 kbp (12). Regulation of viral gene expression is temporally organized relative to the time of viral DNA replication which occurs at approximately 6 hours post infection in cultured insect cells. By our functional criteria, some of the early genes (Table I) are described as immediate early because they are expressed immediately in a viral infection or transient expression assay, or do not require... 

De Sutter V, Vanderhaeghen R, Tilleman S, Lam-mertyn F, Vanhoutte I, Karimi M, Inze D, Goossens A, Hilson P (2005) Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells. Plant 144 1065-1076... 

Figure 2. Effects of ribozyme-expression plasmids specific for cleavage of CBP mRNA on the levels of CBP in a transient-expression assay. Relative levels of CBP were compared by an amplified sandwich ELISA. Normalized levels of CBP in wild-type (WT) F9 cells were taken arbitrarily as 100%. All values are the averages of results from at least three experiments and the standard deviation for each value relative to the value for WT cells is indicated.

To determine the effectiveness of the riboz>me-mediated inhibition of expression of the CBP gene in a transient expression assay, we compared relative levels of CBP by an amplified sandwich ELISA. As shown in Figure 2, all the active ribozymes decreased the level of CBP in vivo. The results... 

An interesting and more recent expression system for the development of cell-based assays is based on BacMams. These recombinant baculoviruses containing mammalian cell-active expression cassettes seem to be an efficient strategy to speed up assay development. These viruses are produced in insect cells and transiently express but do not replicate in transduced mammalian cells. The expression level can be well controlled by titrating the amount of virus. Highly reproducible transient expression levels, which are a prerequisite to use transient transfection for HTS, might thus be an attractive alternative for some approaches. 

Some RNA aptamers that were isolated in vitro have also been expressed in vivo to study their biological function within the context of a living pro- or eucaryotic cell. Among them is an aptamer which binds to the reverse transcriptase (Rev) protein of the human immunodeficiency virus type 1 (HIV-1) [42,43,70],This anti-HIV-l-Rev aptamer was cloned into an expression cassette based on the U6 snRNA promoter, in which aptamer transcripts are protected against nuclease degradation to some extent. Transient expression in the nucleus of cultured cells led to 107-109 full-length aptamer transcripts per cell. When anti-HIV-l-Rev aptamer-expressing cells were co-transfected with the HIV-1 provirus, viral replication was efficiently inhibited in these cells, as shown by an assay in which the production of HIV-1 reverse transcriptase was measured [71],... 

Transient gene expression assays were performed to test whether a functional promoter resided in the 5 -flanking region of exon 5 2. Putative... 

Based on the calponin expression pattern and nucleotide sequence differences it has been established that three genes generate the calponin variants [122,125,128]. Miano and Olson [127] reported that hi calponin transcription begins at two dosely located initiation sites in the promoter. This is probably due to the absence of either a TATAA box or an initiator consensus element, which makes this promoter quite unique compared to those discussed above. In addition, transient transfection assay shows that this promoter is active in cell lines that do not express the endogeneous hi calponin gene. The presence of an upstream polypurine sequence in the promoter might potentially attenuate the level of transcription and, thus, hamper or exclude hi calponin in non-SMC cells [127]. 

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